PROJECT ABSTRACT Alzheimer's disease (AD) is characterized by cholinergic dysfunction and depositions of the amyloid ?-peptide (A?) derived from A? precursor protein (APP). Four current FDA-approved drugs for AD belong to the category of cholinesterase inhibitor (ChEI). These drugs are useful for treatment of mild to moderate AD but limited use in advanced stages. Our goal is to test novel acetylcholinesterase inhibitors (AChEIs) or butyrylcholinesterase inhibitors (BChEIs) against pivotal steps in the pathophysiology of AD to better understand the drugs' effects. Our focus is to test new therapeutic strategies to both validated and novel disease targets. Our hypothesis is that certain ChEIs have neuroprotective activity independent of their cholinergic activity due to their A?-lowering ? property. Our proposal to study the mechanism of ChEI drugs on amyloidogenic pathways is based on our results that certain ChEIs significantly reduced A? levels in cultured cells and animals. The specific aims are: 1] To study the effect of novel AChEIs and BChEIs on APP pathway steps. We will examine i) the effect of novel ChEIs on APP pathway: A? and BACE levels, ii) the effect of substitution of functional groups, iii) APPmRNA 5'-UTR, and iv) synaptic proteins. 2] To test the effect of a novel group of ChEIs on A? deposition in vivo. We will study the effects of different doses of selective AChEIs and BChEIs on APP and A? in double transgenic APPSWE-Tau amyloid plaque plus tangle producing mice. 3] To examine the role of ChE enzyme in AChE knockout mice. We will study the role of the AChE enzyme on APP, A? peptides and synaptic protein markers in the brain tissue samples from novel AChE knockout mice. 4] To investigate the effect of ChEIs on molecular markers in human (archived) samples. To validate the animal studies, we will test the effect of treatment of selected ChEIs on APP and A? levels in human plasma and/or CSF samples. The primary end-points are quantitative and functional: Cell survival, enzyme assay and levels of APP pathway protein/peptides and synaptic protein markers. We will mechanistically select ChEIs that interact with the peripheral allosteric binding domain of ChE and with the esteratic and anionic binding domains and test them in cell culture and animal models. This work will indicate unique effect of ChEIs on A? and synaptic proteins, independent of their selectivity for the enzyme. This property will help maximize their beneficial effects on amyloid and synaptic proteins, which can be utilized to design better therapeutic agents for AD. PROJECT NARRATIVE Alzheimer's disease (AD) is characterized by a reduction in the presynaptic markers of the cholinergic system, particularly in areas of the brain related to memory and learning, and by depositions of the amyloid ?peptide (A?), which is derived from the A? precursor protein (APP). Four of five current FDA-approved drugs for AD are cholinesterase inhibitors (ChEI), which increase the brain's supply of acetylcholine (ACh) by inhibiting cholinesterases (ChE) enzyme and thus preserve cholinergic circuits, which are believed to mediate memory pathways. Rather surprisingly, these drugs did not have a substantial effect on memory, but had an unexpected and welcome outcome of preserving cognition for a slightly extended period of time. The present proposal attempts to characterize the mechanisms of neuropreservation and protection by the ChEIs and fine tune this pathway to improve this beneficial property of ChEIs. Our goal is to test novel acetylcholinesterase inhibitors (AChEIs) or butyrylcholinesterase inhibitors (BChEIs) against pivotal steps in the pathophysiology of AD to better understand the effects of the drugs. We propose to study the mechanism of ChEI drugs on amyloidogenic pathways that process APP to potentially toxic A?. Our studies initially focused on the effects of these drugs on A? production and have recently been extended to synaptic protein markers and other neuroprotective effects. The outcome of the proposal is to identify mechanisms by which ChEIs block potentially toxic A? levels and to utilize this property in developing novel therapeutic agents.